Since its inception1 non-linear fluorescence microscopy (that is, where for a given power input there is non-linear fluorescence intensity), particularly two-photon fluorescence microscopy, has rapidly emerged as an important technique for three-dimensional imaging of biological specimens, and in particular for use in surgical biopsy and early cancer detection. For example in relation to two-photon fluorescence microscopy this rapid emergence can be attributed to advantages offered by two-photon excitation relative to single-photon excitation. These advantages include an inherent optical sectioning property, confinement of photo damage to the focal region and improved depth penetration into a sample1. The feature of confinement arises as two-photon induced absorption is most probable in the focal volume where the photon energy density is highest. In fact, the out-of-focus fluorophores are safeguarded from photobleaching since the energy density is not sufficient to induce fluorescence at these points. The third-order nonlinear excitation probability makes it possible to image a single point of the specimen, preserving the fluorophores at other axial depths for subsequent observation. The ability to discriminate against fluorescence originating from outside of the focal spot is a powerful property of the imaging modality that provides an inherent optical sectioning effect for the acquisition and reconstruction of 3-D images.
However, one of the greatest restrictions to the development of two-photon microscopy particularly in relation to in vivo applications, is that the known apparatus require the use of complicated pulse lasers and bulk optics. While the introduction of optical fibers and fiber components to coherent (single-photon) fluorescence imaging systems2-5 has overcome some physical limitations, and offered an ability to image specimens in vivo by delivering excitation radiation to a remote sample, the present single-photon arrangements remain bulky, cumbersome to operate, expensive and limited in their functionality. In addition to these problems the application of fiber optics to two-photon fluorescence systems has been hindered by the perceived problem of dispersion of the short pulses required for two-photon fluorescence. The present inventors have now devised apparatus that may address or at least obviate to some extent a number of the problems outlined above.
In one aspect of the present invention the microscope or endoscope comprises single-mode fiber for delivery of an ultra short pulsed laser beam, and a multi-port fiber coupler replaces bulk optics for illumination delivery and signal collection. Although a multi-port fiber coupler has been used in scanning differential interference contrast microscopy7, confocal reflection microscopy2,8 and confocal interference microscopy9, such arrangements have not successfully been adopted in relation to two-photon fluorescence microscopy or endoscopy. It was previously thought impractical to adopt such an arrangement due to the fact that the separation of the excitation and fluorescence wavelengths is so large that the fiber coupler would be unable to efficiently transmit light at these distinct wavelengths. However, the present inventors have found that it is possible in the present apparatus for the excitation and fluorescence wavelengths to be transmitted with satisfactory efficiency.
A further advantageous aspect of the present invention is that it exhibits a self-aligning nature, because the illumination delivery and signal collection beams utilise the same port. In embodiments of the invention that utilise a small fiber aperture, image resolution of the new system may be significantly improved, and multiple scattering may be reduced, relative to conventional two-photon fluorescence endoscopes or microscopes (as for example described in UK patent applications 2,353,369 and 2,341,943).
UK patent number 2,338,568 discloses an endoscope/microscope apparatus where the laser light pulses are split by a beam splitter and are reflected back towards the beam splitter by anti-dispersive gratings in order to condense the light pulses. Within this document there is insufficient information provided to demonstrate effectiveness of the arrangement, which is not considered likely to be particularly suitable for use in two-photon fluorescence endoscopic applications due to component bulk and space separation between the dispersing means and sample.
In another aspect the present invention utilises a scanning head for delivery of illuminating light to the sample and for signal collection from the sample. Although a similar approach has previously been used for acquisition of optical coherence tomography (OCT) images of in vitro human saphenous vein10 it was previously considered that this type of micro-scanning head would result in unacceptable levels of temporal dispersion at the increased powers required for non-linear (especially two-photon) fluorescence, compared to those adopted in OTC.